RNAqueous Micro Kit from Life Technologies

March 20, 2013

BlueskyReddit

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
Write a Review
Dept of Research
The Jackson Laboratory
Research Assistant

The image shows an Agilent Bioanalyzer “gel” on which each RNA sample has been run so that you can see the 28S and 18S bands. (There is some degradation in the first sample, so I did not use it for any downstream applications. )

Company:

Life Technologies

Product Name:

RNAqueous Micro Kit

Catalog Number:

AM1931
Image

Our laboratory studies the genetics of hearing loss in mice. When we identify a gene that might be involved in hearing loss, it is often necessary to investigate the expression of that gene using RT-PCR. It is difficult to get good quality RNA from adult inner ears. The yield is usually abysmal. The tissue is so small. Recently we have identified a gene that might be involved in causing hearing loss in mice. It appears that this gene might be important in the development of the inner ear. It became necessary for me to perform RT-PCR using tissue from ears from embryos and early postnatal animals. In light of the fact that it is so difficult to obtain high quality RNA from adult animals, I feared that it would be impossible to get enough RNA from an embryo to perform even a single experiment. A labmate found the RNAqueous Micro Kit. It was advertised as being appropriate for isolating total RNA from cultured cells, laser captured cells, and tissue weighing up to 10 mg.

This kit is very easy to use and can be used to isolate RNA from cultured cells; however, this review discusses isolation of RNA from tissue. In order to isolate RNA from a tissue sample, it is only necessary to homogenize in the lysis buffer which contains guanidinium thiocyanate, then add ethanol and run the sample through the column provided with the kit, wash, and elute.

Experimental Design and Results Summary

Application

RNA isolation for RT-PCR

Starting Material

Inner ears isolated from 14.5 day old mouse embryos.

Protocol Overview

The ears were dissected and flash frozen in liquid nitrogen. I kept them at -80°C until I was ready to isolate the RNA from the tissue. The tissue samples weighed less than 10mg, so I followed the instructions in the manual and used 100ul of lysis solution. I used a mechanical homogenizer to grind up the tissue in the lysis solution. Next, I added 50ul of 100% ethanol and vortexed to mix well. After vortexing, I applied the entire 150ul to the silica column and centrifuged for one minute at maximum speed. I decided to go for a longer spin time of 30 seconds instead of the 10 sec in the protocol to make sure that all of the lysate passed through the filter. It was suggested in the protocol that longer spins might be necessary to filter the lysate from tissue samples. I chose to do a 30 second spin because this was the shortest time on our centrifuge. I washed the filter with 150ul of wash solution 1, which had been mixed with ethanol according to the product instructions prior to beginning the experiment. This wash was followed by a centrifugation step to pass the wash solution through the filter. Two more washes with solutions 2 and 3, which had been mixed with ethanol prior to beginning the experiment, followed the initial wash step. I eluted the RNA in the elution solution that came with the kit. The elution solution must be heated to 75°C prior to use. I performed the elution as suggested in the protocol; I added 5ul of preheated elution solution directly to the column followed by a 30 sec spin and repeated this step. I used a Nanodrop spectrophotometer to check the concentration of these samples.

Tips

As I mentioned above, I chose to increase the initial spin time. While I don’t know if this improved my results, it certainly did not make them any worse. I would certainly recommend it for tissue samples. It can’t hurt. If you are purchasing this kit for the first time, make sure to read the protocol before jumping in to do the experiment. There are certain things like adding ethanol to some of the supplied solutions and heating the elution solution that must be done prior to starting the experiment.

Results Summary

The concentrations of these samples were quite good considering that the original tissue sample size was so small. I was able to obtain concentrations of 200-400ng/ml. The A260/280 ratios for the samples ranged between 1.89 and 2.01, so I was very happy with these results. I also used an Agilent Bioanalyzer to determine the quality of my RNA. All of the RNA that I had isolated using the RNAqueous Micro Kit was of very high quality. All of these samples worked great in my subsequent downstream RT-PCR experiments.

Additional Notes

This kit gave me great results. I would recommend following the instructions in the manual.

Related Categories

Image Gallery

The image shows an Agilent Bioanalyzer “gel” on which each RNA sample has been run so that you can see the 28S and 18S bands. (There is some degradation in the first sample, so I did not use it for any downstream applications. )

Summary

The Good

The RNAqueous Micro Kit makes it very easy to obtain high quality RNA from small tissue samples.

The Bad

I don’t have much bad to say about this kit. I did have a few samples that looked like they were starting to degrade when I ran them on a gel, but overall most of the samples looked good.

The Bottom Line

This kit is fast, easy to use, and gives great results.

Join the discussion